Choline-containing phospholipids serve as precursors for lipid-derived messenger molecules that function as both intra and extracellular signals. This proposal focuses on two pivotal enzymes in this signaling system: phosphatidylcholine-specific phospholipases D (PLD) and phosphatidic acid phosphohydrolases (PAP). Activation of PLD is a widespread reaction of diverse cell types to stimulation with a wide range of agonists and thereby implicated in a broad spectrum of physiological processes including cell growth and differentiation, metabolic regulation and the immune and inflammatory responses. Understanding the regulation and functions of this signaling system could lead to new therapies for treatment of diseases where these processes are aberrantly regulated or inappropriately activated. We have cloned cDNAs encoding mammalian PLD (PLD1 and PLD2 and PLD3) and type-2 PAP enzymes (PAP2a, 2b 2c and 2d). We hypothesize that these enzymes are responsible for synthesis of two lipid mediators: Diacylglycerol (DG) for activation of conventional PKC isoenzymes and the G-protein-coupled receptor ligand lysophosphatidic acid (LPA). We propose a series of experiments to test these hypotheses. Both series of studies share a common design. The potential involvement of the PLD and PAP enzymes in DG and LPA synthesis will be explored by transient transfection of HEK293 cells. Building on the results of these experiments we describe strategies for inhibition or suppression of the endogenously expressed enzymes in model cell lines to determine their normal roles in synthesis of these important lipid mediators.